- Email: [email protected]
Effect of nutrient pulses on photosynthesis of Chaetomorpha linum from a shallow Mediterranean coastal lagoon
Aquatic Botany 82 (2005) 181–192 www.elsevier.com/locate/aquabot
Effect of nutrient pulses on photosynthesis of Chaetomorpha linum from a shallow Mediterranean coastal lagoon Margarita Mene´ndez * Department of Ecology, University of Barcelona, Avgda. Diagonal 645, 08028 Barcelona, Spain Received 29 January 2004; received in revised form 30 March 2005; accepted 7 April 2005
Abstract The influence of nitrogen and phosphorus pulses on Chaetomorpha linum (Muller) Kutzing growth and photosynthesis was studied in laboratory experiments. Photosynthesis and growth of C. linum from Tancada lagoon seems limited by both nitrogen and phosphorus, as indicated by the high rate (4.7–11.6 mg O2 g1 dry weight h1) of light-saturated photosynthesis (Pm) and growth rates observed under nitrogen plus phosphorus enrichment in relation to enrichment by nitrogen alone (2.9–7.6 mg O2 g1 dry weight h1). Significant increase in nitrogen and phosphorus content as percentage of dry weight was observed in C. linum fertilized with a single nutrient or with nitrogen plus phosphorus. In Tancada lagoon, when availability of nitrogen to primary producers is by pulses, an increase of nitrate concentration in the water column (from 6 to 100 mM) has a greater effect on growth of C. linum (growth rate: 0.13 day1) than an increase in ammonium concentration (from 20 to 100 mM and growth rate: 0.11 day1). For a given thallus nitrogen content (0.6–1.4% N), both Pm and the photosynthetic efficiency (a) normalized to dry weight were correlated (r2 = 0.73, p < 0.005) indicating that variations in electron transport were coupled to variations in C-fixation capacity. Optimizing both a and Pm may be a general characteristic of thin-structured opportunistic algae in more variable estuarine environments. # 2005 Elsevier B.V. All rights reserved. Keywords: Chaetomorpha linum; Nitrogen; Phosphorus; Photosynthesis; Growth; Coastal lagoon
* Tel.: +34 93 4021508; fax: +34 93 4111438. E-mail address: [email protected]. 0304-3770/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2005.04.004
182
M. Mene´ndez / Aquatic Botany 82 (2005) 181–192
1. Introduction Light and nitrogen availability are described as the primary factors controlling macroalgal productivity in temperate estuarine waters (McGlathery and Pedersen, 1999). These parameters vary considerably in natural environments owing to nutrient inputs from agricultural or urban activities (McGlathery et al., 1997). Macroalgae living in those environments should have mechanisms that ensure a balance between resource variability and growth. In estuarine environments, nutrient availability may vary as much over short times scales (hours to days) as over seasonal cycles (Ramus and Venable, 1987). Nutrients involved in the enrichment vary among estuaries. In some lagoon environments, the enrichment depends mainly on elevated inputs of dissolved inorganic nitrogen (DIN) (Nixon et al., 1986). In others, the nitrogen loading is accompanied by inputs of phosphorus. In some of these, for example, Buttermilk Bay (Valiela et al., 1990) and Sacca di Goro (Viaroli et al., 1995), most of the DIN enters as nitrate that causes excessive growth of green macroalgae. In others, such as Moriches Bay (Ryther, 1989), DIN enters mainly as ammonium or in organic form, such as urea. In Tancada lagoon (Ebro Delta, north-east Spain), most of the DIN enters as nitrate as a result of fertilization of ricefields in the upland watershed in spring, whereas in autumn–winter, nitrogen enters mainly as ammonium (Mene´ ndez and Comin, 2000) due to decomposition and mineralization of organic matter or by inputs from the adjacent bay (Vidal and Morguı´, 2000; Mene´ ndez unpublished data). Thus, ephemeral macroalgae that grows rapidly and proliferate in estuarine environments must be able to adjust their carbon (C) and nitrogen (N) metabolism and SRP uptake over these short time scales in order to optimize N levels and photosynthetic C fixation. In all plants, the energy and C skeletons required for N uptake and assimilation are provided by photosynthesis, and over 50% of tissue nitrogen is allocated to the chloroplast (Turpin, 1991). Moreover, these opportunistic macroalgae are capable of uptake, assimilation and storage of large amounts of nitrogen in areas of high nitrogen loading resulting in low water column concentrations of nutrients (Peckol et al., 1994). Also, N-deficient green algae accumulate C, mainly as starch reserves, which can later be mobilized by respiration to provide the carbon skeletons necessary for N assimilation when N is resupplied (Vergara et al., 1995; McGlathery and Pedersen, 1999). On the other hand, ephemeral macroalgae growing in estuarine environments are exposed to variable irradiance, mainly in shallow environments frequently affected by strong winds, such as Tancada coastal lagoon. It was also reported that the mechanism of light acclimation is related to the algal nitrogen status (McGlathery et al., 1996). This study aims to analyse the effects of different nutrient pulses on Chaetomorpha linum photosynthetic capacity and growth from Tancada lagoon, which is affected by high fluctuations of water inputs. These fluctuations are both in flow rates and in water quality, which can be freshwater from ricefields or seawater from the adjacent bay. Laboratory experiments were conducted to compare the effects of different nutrient fertilization on photosynthetic efficiency at low irradiance (a) and to determine the maximum rate of photosynthesis at saturating irradiance (Pm) and respiration (R).
M. Mene´ ndez / Aquatic Botany 82 (2005) 181–192
183
2. Materials and methods 2.1. Study area Tancada is a small, shallow coastal lagoon located in the Ebro River Delta, NE Spain (408400 N, 08360 E, area of 1.8 km2, average depth of 37 cm). This lagoon receives freshwater from irrigated ricefields in spring and summer, mainly in the west basin which has a relatively high concentration of nitrates in April (about 117 mM). During late spring and summer, DIN concentration in water column remains between 0 and 1 mM due to uptake by primary producers, mainly floating macroalgae (C. linum, Cladophora, Gracilaria verrucosa, Ulva) and by the aquatic phanerogam Ruppia cirrhosa. In autumn and winter, ammonium concentration increases, mainly in the east basin (about 40 mM). Soluble reactive phosphorus (SRP) concentrations in the lagoon were normally low (between 0.05 and 5.09 mM), principally due to the adsorption process into particles being partly irreversible, so that a fraction of phosphorus is ultimately lost in the sediments (Vidal, 1994). Minimum SRP concentrations were observed in spring and summer and maximum in autumn and winter, with sporadic values of 8.3–12.8 mM in autumn when strong winds are frequent. Water conductivity ranges from 12 to 16 mS cm1 in the west basin and between 14 and 42 mS cm1 in the east basin. The dissolved inorganic carbon (DIC) concentration in the water column varies between 3.0 and 3.9 mmol L1, and the pH remains fairly constant around 8.2 (Mene´ ndez et al., 2001). 2.2. Experimental design C. linum (Mu¨ ller) Ku¨ tzing was collected at the end of May 1996 from Tancada lagoon. Approximately 10 g (fresh weight) of macroalgae were placed in 2 L glass jars filled with filtered (Watman GF/C) water from the lagoon and were allowed to acclimate for 3 days before the experiment. Background levels of nutrients in the incubation water were <2 mM inorganic phosphorus, <20 mM NH4+ and <6 mM NO3. Circulation of water in the recipient was provided by bubbling with compressed air. The experiment was conducted in a temperature controlled room (22–23 8C) on a 15:9 h light–dark cycle, with light provided by fluorescent (400 W) lamps at a level of 400 mmol photons m2 s1, which saturates C. linum photosynthesis (McGlathery et al., 1996; Mene´ ndez and Comin, 2000). Water conductivity in the jars was maintained at arround 40–41 mS cm1. The experiment consisted of six treatments, each conducted in triplicate. Three recipients received additions of PO43; three received additions of NO3; three received NH4+; three received NO3 + PO43; three received NH4+ + PO43; and three recipients were not enriched, serving as controls (CT). The nutrients were added in dissolved form from stock solutions of KH2PO4, NaNO3, and NH4Cl to the final concentrations of 17 mM PO43, 100 mM NH4+ and 100 mM NO3, respectively. Nutrient additions were made at days 0 and 4 of the experiment. Macroalgae were collected 9 days after the experiment began. Subsamples of macroalgal biomasses used as the inoculum (day 0) and those from each treatment (day 9) were collected and used for chlorophylls, dry weight, C, N and P analyses. chlorophyll a and b concentrations were determined on fresh thalli spectrophotometrically from 90%
M. Mene´ ndez / Aquatic Botany 82 (2005) 181–192
184
(v/v) acetone extracts as described by Strickland and Parsons (1972). Dry weight at 60 8C (to constant weight) and total carbon and nitrogen (determined as percentage of sample dry weight on duplicate finely ground samples using a Carlo-Erba analyzer) were analyzed from the algae tissue. Total P content was determined after acid digestion by colorimetry (Jackson, 1970). The net growth rate (m) of C. linum was calculated following the equation: m = (ln Btln B0)t1, where Bt and B0 are the biomasses (dry weight) of the algae at the end and the beginning of the experiment, respectively, and t is the number of days. 2.3. Photosynthesis versus irradiance measurements In previous studies, we have observed that after 15 and 60 h of fertilization with phosphorus and nitrogen, respectively, almost all the dissolved inorganic phosphorus (from 17 to 0.8 mM) and nitrogen (from 100 to 15 mM) added to the jars with C. linum was absorbed (Mene´ ndez et al., 2002). Photosynthesis and dark respiration of C. linum were measured three times: (1) 3 days after the first fertilization, during the depletion period to measure the effect of nutrient storage in macroalgae; (2) 3 h after the second fertilization; and (3) 1.5 days after the second fertilization. In control jars, photosynthesis and respiration were measured on the same days as the rest of the treatments, but owing that were not fertilized, measurements corresponding to 3, 4 and 4.5 days without fertilization. Measurements were made as oxygen changed in filtered (0.45 mm mesh size) lagoon water (initial pH 8.94 0.029 and DIC 3.01 0.0046 mmol L1) in 110 mL stoppered bottles containing about 0.5 g fresh weight (0.03 g dry weight) of whole algal thalli (McGlathery et al., 1996). The bottles were placed in an incubator at a controlled temperature (20 8C), and stirring was performed by stirring bars (ca. 200 rpm). Daylight fluorescent tubes were used and the irradiance was adjusted by covering the bottles with neutral filters to assess photosynthesis–irradiance relationships (between 0 and 830 mmol photon m2 s1, 400– 700 nm). After 1.5 h of incubation, dissolved oxygen concentrations were measured to the nearest 0.01 mg using an oxygen electrode (MultiLine P3 WTW instruments) and checked with oxygen concentrations in bottles (n = 3) without algae. The alga was extracted of each bottle and was dried to constant weight at 70 8C. All the results were expressed in mg O2 g1 dry weight h1. Parameters describing the photosynthesis versus irradiance response (P–I) were determined for each experimental condition using a Sigmaplot computer program by fitting the observed data to the equation (Webb et al., 1974): P ¼ Pm ð1 expaI Þ þ R where P is gross photosynythesis, Pm is the maximum rate of net photosynthesis, a is initial slope representing photosynthetic efficiency at low light, I is irradiance, and R is respiration rate. 2.4. Statistical analysis The effects of nutrient enrichments on nutrient and chlorophyll content of the algae and on growth rate were statistically tested using one-way ANOVA. Data were log transformed
M. Mene´ ndez / Aquatic Botany 82 (2005) 181–192
185
Fig. 1. Nitrogen and phosphorus contents as percentage of dry weight in C. linum 0 day (represented by lines) and at 9 days (represented by bars) of the incubation period. Mean of three replicates and standard errors are reported. An asterisk represents significant differences with the control and between the beginning and the end of the experiment at the p < 0.05 level.
to obtain homogeneity of variances and determined to be normally distributed (Lilliefors test) before the analyses were conducted. Post hoc comparisons were made among treatment means using Tukey’s test, when ANOVA demonstrated that significant differences existed. To test the effect of enrichment on P–I curves, Student’s (paired) t-test (Legendre and Legendre, 1998) were used with a level of significance at 5%. The CSSStatistica computer program was used for statistical analysis.
3. Results 3.1. Algal content of N, P and C Enrichment with N alone, or in combination with P, resulted in significant increase (F = 53.8, Tukey’s test, p < 0.0005) of the N content of C. linum (Fig. 1), while enrichment with P alone had no effect (Tukey’s test, p > 0.05) on tissue-N. Enrichment with P alone or in combination with N, resulted in significant (F = 93.36, Tukey’s test, p < 0.0005) increases in the P content of C. linum, and this increase was higher in the treatments fertilized with P alone than in those treatments where both P and N, either as nitrate or ammonium, were added (Fig. 1). N-enrichments alone had no effect (Tukey’s test, p > 0.05) on the P content of C. linum. N enrichment increased the N:P ratio up to 21 in both nitrate plus P, and ammonium plus P treatments, and to 192–194 under N-alone enrichments as ammonium or nitrate related to the P-alone treatment (Table 1). A significant increase in carbon content was observed in the tissue of C. linum in the control jars (no nutrient additions) after 9 days (F = 3.05, Tukey’s test, p < 0.05). 3.2. Chlorophyll content of the algae N-enrichment was followed by an increase in the chlorophyll a and b content in the tissue of algae (F = 5.87, p < 0.01), and this increase was higher in the jars enriched with
186
M. Mene´ ndez / Aquatic Botany 82 (2005) 181–192
Table 1 Atomic N:P, C:N, C:P ratios and growth rates (m, day1) calculated from C. linum between 0 and 9 days
Initial NO3 NH4+ PO43 NH4+ + PO43 NO3 + PO43 Control
N:P ratio
C:N ratio
C:P ratio
m (day1)
63 0.2 194 11.4 192 53.5 6 0.9 21 4.2 21 0.2 82 0.2
32.9 2.15 19.7 0.03 19.5 0.79 41.8 5.16 19.6 0.63 20.9 1.83 47.6 0.01
3141 32 3780 46 4545 89 249 20 493 11 437 21 3809 40
– 0.130 0.002 0.114 0.004 0.101 0.001 0.110 0.007 0.128 0.007 0.088 0.001
Values are the mean calculated from algae in the three recipients used for each treatment (S.E.)
ammonium alone than in those enriched with nitrate alone or N, either ammonium or nitrate, plus P (Fig. 2). The chlorophyll a/b ratio was significantly lower in C. linum fertilized with ammonium plus P than in control jars. 3.3. Growth rates An increase of biomass was observed in all the treatments after 9 days of the experiment; the growth rate of C. linum was significantly higher in enriched jars than in controls (F = 12.15, p < 0.001) (Table 1). No significant differences were observed in growth rates between different fertilization treatments, except between C. linum enriched with nitrate and nitrate plus P related to growth rate obtained in P-alone treatment (Tukey’s test, p < 0.05). 3.4. Photosynthesis nutrients relationships A significant effect was observed in C. linum photosynthesis under N fertilization, both when it was added alone and in combination with P in the incubation performed after the second fertilization (t > 2.5, p < 0.05, eight comparisons: all treatments with nitrogen versus CT, after 3 h and 1.5 days). No significant differences were observed
Fig. 2. Concentration of total (a + b) chlorophyll and chlorophyll a/b ratios in C. linum at the end of the experiment (9 days). Mean of three replicates and standard errors are reported. An asterisk represents significant differences with the control at the p < 0.05 level.
M. Mene´ ndez / Aquatic Botany 82 (2005) 181–192
187
between incubations made after 3 h and 1.5 days of fertilization with both types of N alone, but the photosynthetic response was higher when nitrogen was added as ammonium than as nitrate (t > 2.2, p < 0.05, two comparisons: ammonium versus nitrate alone treatments after 3 h and 1.5 days). If N was added in combination with P, significant differences in photosynthesis were observed after 3 h and 1.5 days of fertilization when N was in the form of ammonium (t = 3.8, p < 0.005, one comparison: ammonium plus P after 3 h versus 1.5 days), but not when N was added as nitrate. Inhibition of the photosynthesis was observed only in the incubation made with C. linum during the depletion period after 3 days of the first nitrate plus P fertilization. The maximum value of Pm (11.6 mg O2 g1 dry weight h1) was observed after 3 h of fertilization with ammonium plus P (Table 2). No significant differences in photosynthesis were observed between enrichment treatments and controls during the depletion period (after 3 days of the first fertilization) (Table 2). Addition of P had a significant effect increasing the photosynthesis after 1.5 days of the second fertilization in relation with the control after 4.5 days (3 + 1.5 days) without P addition (t > 3.6, p < 0.005). In the jars without enrichment (CT jars), a significant decrease in photosynthesis was observed after 5 days (t > 5.5, p < 0.0005, two comparisons: CT after 3 and 4 days versus CT after 4.5 days). The addition of nitrate alone increased C. linum respiration after 3 h of fertilization, but this effect was not observed after 1.5 days (Table 2). Increasing values of respiration were observed in C. linum enriched with P alone or in combination with nitrate or ammonium; this effect was higher when N was added as ammonium. Fertilization with ammonium alone had no effect on respiration rate. Nutrient enhancement of photosynthetic efficiency under low light (a) was observed in all the jars fertilized. This increase was higher under ammonium plus P treatment (Table 2). An inverse relationship was observed between a and chlorophyll a/b ratio (r2 = 0.86, p < 0.05) (Fig. 3a).
4. Discussion From the results obtained in this experiments, both photosynthesis and growth of C. linum from Tancada lagoon were limited by N and P, because both added nutrients, alone or in combination, enhanced the Pm and growth rates. In fact, considering both Pm and growth rates, one finds that N has a major effect with respect to P. The atomic N:P ratio of the algae in the treatments with P alone (6.0) was below the ratio of 36 reported by Atkinson and Smith (1983), indicating deficiency of N relative to P. The C:P ratio was five to eight times above the value of 550 (Atkinson and Smith, 1983) in all the treatments without P, showing deficiency of P (Lapointe, 1997). However, availability of N and P would be very variable along all the annual cycle in the field; consequently physiological and morphological responses of the macroalgae could be very different under different environmental conditions. The growth rate ranged between 0.087 and 0.130 day1, which is within the range of growth rates reported for C. linum measured in laboratory (0.03–0.15 day1; McGlathery and Pedersen, 1999) and in situ (0.01–0.22 day1; Pedersen and Borum,
188
NH4+ NO3 PO43 NH4+ + PO43 NO3 + PO43 CT a b
Pm (mg O2 g1 dry weight h1)
R (mg O2 g1 dry weight h1)
3 days
3h
1.5 days
3 days
2.96 3.27 2.38 6.90 4.72 2.33
7.19 6.08 3.11 11.61 7.48 2.71
7.66 5.48 3.88 9.78 6.25 1.53
0.73 0.92 0.66 0.85 0.62 0.25
(0.34) (0.47) (0.24) (0.85) (1.14) (0.39)
(0.71) (0.54) (0.45) (1.01) (0.55) (0.49)a
4 days after the beginning of the experiment. 4.5 days after the beginning of the experiment.
(0.79) (0.64) (0.60) (0.75) (0.71) (0.21)b
3h (0.18) (0.23) (0.13) (0.47) (0.32) (0.21)
0.82 1.34 1.01 0.44 1.36 0.23
(0.38) (0.30) (0.23) (0.34) (0.30) (0.17)a
a (mg O2 g1 dry weight h1/mmol m2 s1) 1.5 days
3 days
3h
0.76 0.66 1.33 1.72 1.01 0.85
0.020 0.017 0.010 0.020 0.014 0.014
0.038 0.026 0.012 0.036 0.041 0.009
(0.42) (0.35) (0.33) (0.39) (0.38) (0.11)b
(0.0025) (0.0033) (0.0019) (0.0025) (0.0060) (0.0037)
1.5 days (0.0023) (0.0018) (0.0022) (0.0014) (0.0018) (0.0046)a
0.039 0.033 0.025 0.064 0.037 0.009
(0.0019) (0.0026) (0.0044) (0.0015) (0.0036) (0.0039)b
M. Mene´ ndez / Aquatic Botany 82 (2005) 181–192
Table 2 Maximum photosynthetic rate (Pm), respiration (R) and photosynthetic efficiency at low irradiances (a), and standard errors in parenthesis, estimated from the experimental incubations of C. linum with different nutrient additions, 3 days after the first fertilization (depletion period) and 3 h and 1.5 days after the second fertilization (see Section 2)
M. Mene´ ndez / Aquatic Botany 82 (2005) 181–192
189
Fig. 3. Relationship between Chlorophyll a/b ratio and a at the end of the experiment (a), and relationship between maximum photosynthetic rate (Pm) and photosynthetic efficiency under low light (a) over the range of tissue N levels (b).
1997). Some discrepancies were observed between growth rates and Pm. The highest photosynthetic rates were observed with ammonium plus P fertilization, but the highest growth rates occurred with nitrate and nitrate plus P fertilization. N is considered to be a limiting nutrient for primary production in temperate coastal ecosystems (Lobban and Harrison, 1994). Nitrate uptake occurs principally during the dark period, whereas, nitrate reductase activity is greatest during the day (Pedersen and Borum, 1997; Lopes et al., 1997). In this study, the nutrients were added during the light period, and most likely, uptake of ammonium was faster than nitrate uptake under light conditions. So, the results observed may be related to the fact that Pm is an instantaneous value of production in a short period of time (1.5 h) with light conditions, and growth rate reflected integrated production throughout the study (9 days), which incubated alternating light and dark conditions. Another explanation is that the cause of the larger Pm in the ammonium treatment results from a greater chlorophyll content which is consistent with the larger value for a in the ammonium compared to the nitrate treatment.
190
M. Mene´ ndez / Aquatic Botany 82 (2005) 181–192
4.1. Effect of enrichment in photosynthesis and chlorophyll concentration Increase of Pm in C. linum occurred rapidly in response to variations in the external N supply, but some differences were observed under treatments. These differences could be related to the fast utilization of P when N is available (Lapointe, 1997). In a previous study, after 15 h of C. linum fertilization (17 mM PO43), all the phosphorus dissolved in the water was absorbed (Mene´ ndez et al., 2002). Moreover, the highest a value (0.064 mg O2 g1 dry weight h1/mmol m2 s1) was obtained in the ammonium plus P treatment after 1.5 days of fertilization, suggesting a high efficiency of light use. In our study, inhibition of photosynthesis at light intensities higher than 300 mmol m2 s1 was observed only with nitrate plus P fertilization during the depletion period (after 3 days of the first fertilization). Photosynthesis may be inhibited by increased oxygen concentration, dissolved inorganic carbon depletion, and high pH and high light intensities (Falkowski and Raven, 1997). The absence of photosynthesis inhibition under ammonium plus phosphorus, in the same conditions that in the nitrate plus phosphorus experiments (pH variation from 8.96 to 9.56, 6% of DIC reduction in 1.5 h and the same irradiances), suggests that this may be in relation with the assimilation of nitrate stored in macroalgae by reduction catalized by nitrate reductase. In addition to carbon products, algal growth required reduced nitrogen (Behrenfeld et al., 2004). If based on nitrate, assimilation to an a-amino acid requires 10 electrons and one ATP that consumes a fraction of photosynthetic reductants associated with stimulated respiratory CO2 release in the light (Weger and Turpin, 1989). In this study, respiration rates increased immediately, following the addition of N, and this effect was more evident when N was added as nitrate. Probably, this increase in respiration rate is in relation to the reduction of nitrate and also in relation to supply of the carbon skeletons for protein synthesis (Turpin, 1991). N deficiency typically causes decrease in both photosynthesis pigments and Rubisco content/activity that correspond to marked declines in a (Falkowski and Owens, 1980) and Pm normalized to biomass (Turpin, 1991). In our study, a significant increase of chlorophyll was observed after N enrichment, and this effect was higher with ammonium enrichment showing N chlorophyll storage. Ammonium has a fast turnover rate into the algal cells, but little importance as a storage pool (McGlathery et al., 1996), whereas nitrate is an important nitrogen reserve pool in green macroalgae (Lopes et al., 1997; Naldi and Wheeler, 1999). This is probably the reason for a low increase in photosynthetic pigments when nitrate was added instead of ammonium as a source of N. Light-saturated photosynthesis and a (normalized to dry weight) were positively correlated over the range of tissue N levels (0.6–1.4% dry weight) (Fig. 3b), indicating that chlorophyll and Rubisco were equally limiting to photosynthesis, and that, light harvesting and C fixation were coupled (Falkowski and Owens, 1980). Photosynthetic efficiency at low irradiance (a) is regulated mainly by the effect of pigment level and configuration on light absorption and electron transport capacities (Turpin, 1991). In our study, an inverse relationship was observed between a and chlorophyll a/b ratio (Fig. 3a), showing that, when C. linum was acclimated to low irradiances (high values of a), a relatively high concentration of chlorophyll b related to chlorophyll a was observed. This structural modification leads to the absorption of longer wavelengths and allow the macroalgae to be more efficient under low light conditions.
M. Mene´ ndez / Aquatic Botany 82 (2005) 181–192
191
McGlathery and Pedersen (1999) suggested that a strategy to optimize both a and Pm may be a general characteristic of opportunistic thin-structured algae, such as C. linum, that represents a compromise between increasing light-harvesting and carbon-fixation capacities when the algae is exposed to variable light and nutrient regimes in estuarine environments. Correlations between a and Pm have been observed in Mediterranean marine macrophytes (Enriquez et al., 1996), macroalgae (Henley et al., 1991) and phytoplankton (Behrenfeld et al., 2004). This would be very beneficial for macroalgal growth in shallow coastal aquatic ecosystems, characterised by frequent wind events and nutrient pulsing discharges, that brings to high variability in irradiance and availability of nutrients.
Acknowledgements This work was supported by the research project ENV4-CT97-0584. European Commission.
References Atkinson, M.J., Smith, S.V., 1983. C:N:P ratios of benthic marine plants. Limnol. Oceanogr. 28, 567–574. Behrenfeld, M.J., Prasil, O., Babin, M., Bruyant, F., 2004. In search of a physiological basis for covariations in light-limited and light-saturated photosynthesis. J. Phycol. 40, 4–25. Enriquez, S., Duarte, C.M., Sand-Jensen, K., Nielsen, S.L., 1996. Broad-scale comparison of photosynthetic rates a cross phototrophic organisms. Oecology 108, 197–206. Falkowski, P.G., Owens, T.G., 1980. Light-shade adaptation: two strategies in marine phytoplankton. Plant Physiol. 66, 592–595. Falkowski, P.G., Raven, J.A., 1997. Aquatic Photosynthesis. Blackwell Science, Massachusetts, 375 pp. Henley, W.J., Levavasseur, G., Franklin, L.A., Osmond, C.B., Ramus, J., 1991. Photoacclimation and photoinhibition in Ulva rotundata as influenced by nitrogen availability. Planta 184, 235–243. Jackson, M.L., 1970. Ana´ lisis quı´mico del suelo. Omega, Barcelona, 662 pp. Lapointe, B.E., 1997. Nutrient theresholds for bottom-up control of macroalgal blooms on coral reefs in Jamaica and southeast Florida. Limnol. Oceanogr. 42, 1119–1131. Legendre, P., Legendre, L., 1998. Numerical Ecology, second ed. Elsevier, Amsterdam, 853 pp. Lobban, C.S., Harrison, P.J., 1994. Seaweed Ecology and Physiology. Cambridge University Press, Cambridge, 366 pp. Lopes, P., Oliveira, M.C., Colepicolo, P., 1997. Diurnal fluctuation of nitrate reductase activity in the marine red alga Gracilaria tenuistipitata (Rhodophyta). J. Phycol. 33, 225–231. Mene´ ndez, M., Comin, F.A., 2000. Spring and summer proliferation of floating macroalgae in a Mediterranean coastal lagoon (Tancada lagoon, Ebro Delta, NE Spain). Est. Coast. Shelf Sci. 51, 215–226. Mene´ ndez, M., Martı´nez, M., Comı´n, F.A., 2001. A comparative study of the effect of pH and inorganic carbon resources on the photosynthesis of three floating macroalgae species of a Mediterranean coastal lagoon. J. Exp. Mar. Biol. Ecol. 256 (1), 123–136. Mene´ ndez, M., Herrera, J., Comı´n, F.A., 2002. Effect of nitrogen and phosphorus supply on growth, chlorophyll content and tissue composition on the macroalga Chaetomorpha linum (O.F. Mu¨ ll) Ku¨ tz in a Mediterranean coastal lagoon. Sci. Mar. 66, 355–364. McGlathery, K.J., Pedersen, M.F., 1999. The effect of growth irradiance on the coupling of carbon and nitrogen metabolism in Chaetomorpha linum (Chlorophyta). J. Phycol. 35, 721–731. McGlathery, K.J., Pedersen, M.F., Borum, J., 1996. Changes in intracellular nitrogen pools and feedback controls on nitrogen uptake in Chaetomorpha linum (Chlorophyta). J. Phycol. 32, 393–401.
192
M. Mene´ndez / Aquatic Botany 82 (2005) 181–192
McGlathery, K.J., Krause-Jensen, D., Rysgaard, S., Christensen, P.B., 1997. Patterns of ammonium uptake within dense mats of the filamentous macroalga Chaetomorpha linum. Aquat. Bot. 59, 99–115. Naldi, M., Wheeler, P.A., 1999. Changes in nitrogen pools in Ulva fenestrata (Chlorophyta) and Gracilaria pacifica (Rhodophyta) under nitrate and ammonium enrichment. J. Phycol. 35, 70–77. Nixon, S.W., Owiat, C.A., Frithsen, J., Sullivan, B., 1986. Nutrients and the productivity of estuarine and coastal marine systems. J. Limnol. Soc. S. Afr. 12, 43–71. Peckol, P., DeMeo-Anderson, B., Rivers, J., Valiela, I., Maldonado, M., Weiner, J., Yates, J., 1994. Growth, nutrient uptake capacities, and tissue constituents of the macroalgae, Gracilaria tikvahiae and Cladophora vagabunda, related to site-specific nitrogen loading rates. Mar. Biol. 121, 175–185. Pedersen, M.F., Borum, J., 1997. Nutrient control of estuarine macroalgae: growth strategy and the balance between nitrogen requirements and uptake. Mar. Ecol. Prog. Ser. 161, 155–163. Ramus, J., Venable, M., 1987. Temporal ammonium patchiness and growth rate in Codium and Ulva (Ulvophyceae). J. Phycol. 23, 518–523. Ryther, J.H., 1989. Historical perspective of phytoplankton blooms on Long Island and the green tides of the 1950s. In: Cosper, E.M., Bricelj, V.M., Carpenter, E.J. (Eds.), Novel Phytoplankton Blooms. Coastal and Estuarine Studies Springer-Verlag, Berlı´n, pp. 375–381. Strickland, J.D., Parsons, T.R., 1972. A Practical Handbook of Seawater Analysis. Bulletin 167, second ed. Fisheries Research Board of Canada, Ottawa. Turpin, D.H., 1991. Effects of inorganic N availability on algal photosynthesis and carbon metabolism. J. Phycol. 27, 14–20. Valiela, I., Costa, J., Foreman, K., Teal, J.M., Howes, B., Aubrey, B., 1990. Transport of groundwater-borne nutrients from watersheds and their effects on coastal waters. Biogeochemistry 10, 177–197. Vergara, J.J., Bird, K.T., Niell, F.X., 1995. Nitrogen assimilation following ammonium pulses in the red algae Gracilariopsis lemaneiformis: effect on C metabolism. Mar. Ecol. Prog. Ser. 122, 253–263. Viaroli, P., Bartoli, M., Bondavalli, C., Naldi, M., 1995. Oxygen fluxes and dystrophy in a coastal lagoon colonized by Ulva rigida (Sacca di Goro, Po River Delta, northern Italy). Fresenius Environ. Bull. 4, 381–386. Vidal, M., 1994. Phosphate dynamics tied to sediment disturbances in Alfacs Bay (NW Mediterranean). Mar. Ecol. Prog. Ser. 110, 211–221. Vidal, M., Morguı´, J.A., 2000. Close and delayed benthic–pelagic coupling in coastal ecosystems: the role of physical constraints. Hydrobiology 429, 105–113. Webb, W.L., Newton, M., Starr, D., 1974. Carbon dioxide exchange of Alnus rubra: a mathematical model. Oecology 17, 281–291. Weger, H.G., Turpin, D.H., 1989. Mitochondrial respiration can support NO3 and NO2 reduction during photosynthesis, respiration, and N assimilationin the N-limited green algae Selenastrum minutum. Plant Physiol. 64, 828–832.
Effect of nutrient pulses on photosynthesis of Chaetomorpha linum from a shallow Mediterranean coastal lagoon Margarita Mene´ndez * Department of Ecology, University of Barcelona, Avgda. Diagonal 645, 08028 Barcelona, Spain Received 29 January 2004; received in revised form 30 March 2005; accepted 7 April 2005
Abstract The influence of nitrogen and phosphorus pulses on Chaetomorpha linum (Muller) Kutzing growth and photosynthesis was studied in laboratory experiments. Photosynthesis and growth of C. linum from Tancada lagoon seems limited by both nitrogen and phosphorus, as indicated by the high rate (4.7–11.6 mg O2 g1 dry weight h1) of light-saturated photosynthesis (Pm) and growth rates observed under nitrogen plus phosphorus enrichment in relation to enrichment by nitrogen alone (2.9–7.6 mg O2 g1 dry weight h1). Significant increase in nitrogen and phosphorus content as percentage of dry weight was observed in C. linum fertilized with a single nutrient or with nitrogen plus phosphorus. In Tancada lagoon, when availability of nitrogen to primary producers is by pulses, an increase of nitrate concentration in the water column (from 6 to 100 mM) has a greater effect on growth of C. linum (growth rate: 0.13 day1) than an increase in ammonium concentration (from 20 to 100 mM and growth rate: 0.11 day1). For a given thallus nitrogen content (0.6–1.4% N), both Pm and the photosynthetic efficiency (a) normalized to dry weight were correlated (r2 = 0.73, p < 0.005) indicating that variations in electron transport were coupled to variations in C-fixation capacity. Optimizing both a and Pm may be a general characteristic of thin-structured opportunistic algae in more variable estuarine environments. # 2005 Elsevier B.V. All rights reserved. Keywords: Chaetomorpha linum; Nitrogen; Phosphorus; Photosynthesis; Growth; Coastal lagoon
* Tel.: +34 93 4021508; fax: +34 93 4111438. E-mail address: [email protected]. 0304-3770/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2005.04.004
182
M. Mene´ndez / Aquatic Botany 82 (2005) 181–192
1. Introduction Light and nitrogen availability are described as the primary factors controlling macroalgal productivity in temperate estuarine waters (McGlathery and Pedersen, 1999). These parameters vary considerably in natural environments owing to nutrient inputs from agricultural or urban activities (McGlathery et al., 1997). Macroalgae living in those environments should have mechanisms that ensure a balance between resource variability and growth. In estuarine environments, nutrient availability may vary as much over short times scales (hours to days) as over seasonal cycles (Ramus and Venable, 1987). Nutrients involved in the enrichment vary among estuaries. In some lagoon environments, the enrichment depends mainly on elevated inputs of dissolved inorganic nitrogen (DIN) (Nixon et al., 1986). In others, the nitrogen loading is accompanied by inputs of phosphorus. In some of these, for example, Buttermilk Bay (Valiela et al., 1990) and Sacca di Goro (Viaroli et al., 1995), most of the DIN enters as nitrate that causes excessive growth of green macroalgae. In others, such as Moriches Bay (Ryther, 1989), DIN enters mainly as ammonium or in organic form, such as urea. In Tancada lagoon (Ebro Delta, north-east Spain), most of the DIN enters as nitrate as a result of fertilization of ricefields in the upland watershed in spring, whereas in autumn–winter, nitrogen enters mainly as ammonium (Mene´ ndez and Comin, 2000) due to decomposition and mineralization of organic matter or by inputs from the adjacent bay (Vidal and Morguı´, 2000; Mene´ ndez unpublished data). Thus, ephemeral macroalgae that grows rapidly and proliferate in estuarine environments must be able to adjust their carbon (C) and nitrogen (N) metabolism and SRP uptake over these short time scales in order to optimize N levels and photosynthetic C fixation. In all plants, the energy and C skeletons required for N uptake and assimilation are provided by photosynthesis, and over 50% of tissue nitrogen is allocated to the chloroplast (Turpin, 1991). Moreover, these opportunistic macroalgae are capable of uptake, assimilation and storage of large amounts of nitrogen in areas of high nitrogen loading resulting in low water column concentrations of nutrients (Peckol et al., 1994). Also, N-deficient green algae accumulate C, mainly as starch reserves, which can later be mobilized by respiration to provide the carbon skeletons necessary for N assimilation when N is resupplied (Vergara et al., 1995; McGlathery and Pedersen, 1999). On the other hand, ephemeral macroalgae growing in estuarine environments are exposed to variable irradiance, mainly in shallow environments frequently affected by strong winds, such as Tancada coastal lagoon. It was also reported that the mechanism of light acclimation is related to the algal nitrogen status (McGlathery et al., 1996). This study aims to analyse the effects of different nutrient pulses on Chaetomorpha linum photosynthetic capacity and growth from Tancada lagoon, which is affected by high fluctuations of water inputs. These fluctuations are both in flow rates and in water quality, which can be freshwater from ricefields or seawater from the adjacent bay. Laboratory experiments were conducted to compare the effects of different nutrient fertilization on photosynthetic efficiency at low irradiance (a) and to determine the maximum rate of photosynthesis at saturating irradiance (Pm) and respiration (R).
M. Mene´ ndez / Aquatic Botany 82 (2005) 181–192
183
2. Materials and methods 2.1. Study area Tancada is a small, shallow coastal lagoon located in the Ebro River Delta, NE Spain (408400 N, 08360 E, area of 1.8 km2, average depth of 37 cm). This lagoon receives freshwater from irrigated ricefields in spring and summer, mainly in the west basin which has a relatively high concentration of nitrates in April (about 117 mM). During late spring and summer, DIN concentration in water column remains between 0 and 1 mM due to uptake by primary producers, mainly floating macroalgae (C. linum, Cladophora, Gracilaria verrucosa, Ulva) and by the aquatic phanerogam Ruppia cirrhosa. In autumn and winter, ammonium concentration increases, mainly in the east basin (about 40 mM). Soluble reactive phosphorus (SRP) concentrations in the lagoon were normally low (between 0.05 and 5.09 mM), principally due to the adsorption process into particles being partly irreversible, so that a fraction of phosphorus is ultimately lost in the sediments (Vidal, 1994). Minimum SRP concentrations were observed in spring and summer and maximum in autumn and winter, with sporadic values of 8.3–12.8 mM in autumn when strong winds are frequent. Water conductivity ranges from 12 to 16 mS cm1 in the west basin and between 14 and 42 mS cm1 in the east basin. The dissolved inorganic carbon (DIC) concentration in the water column varies between 3.0 and 3.9 mmol L1, and the pH remains fairly constant around 8.2 (Mene´ ndez et al., 2001). 2.2. Experimental design C. linum (Mu¨ ller) Ku¨ tzing was collected at the end of May 1996 from Tancada lagoon. Approximately 10 g (fresh weight) of macroalgae were placed in 2 L glass jars filled with filtered (Watman GF/C) water from the lagoon and were allowed to acclimate for 3 days before the experiment. Background levels of nutrients in the incubation water were <2 mM inorganic phosphorus, <20 mM NH4+ and <6 mM NO3. Circulation of water in the recipient was provided by bubbling with compressed air. The experiment was conducted in a temperature controlled room (22–23 8C) on a 15:9 h light–dark cycle, with light provided by fluorescent (400 W) lamps at a level of 400 mmol photons m2 s1, which saturates C. linum photosynthesis (McGlathery et al., 1996; Mene´ ndez and Comin, 2000). Water conductivity in the jars was maintained at arround 40–41 mS cm1. The experiment consisted of six treatments, each conducted in triplicate. Three recipients received additions of PO43; three received additions of NO3; three received NH4+; three received NO3 + PO43; three received NH4+ + PO43; and three recipients were not enriched, serving as controls (CT). The nutrients were added in dissolved form from stock solutions of KH2PO4, NaNO3, and NH4Cl to the final concentrations of 17 mM PO43, 100 mM NH4+ and 100 mM NO3, respectively. Nutrient additions were made at days 0 and 4 of the experiment. Macroalgae were collected 9 days after the experiment began. Subsamples of macroalgal biomasses used as the inoculum (day 0) and those from each treatment (day 9) were collected and used for chlorophylls, dry weight, C, N and P analyses. chlorophyll a and b concentrations were determined on fresh thalli spectrophotometrically from 90%
M. Mene´ ndez / Aquatic Botany 82 (2005) 181–192
184
(v/v) acetone extracts as described by Strickland and Parsons (1972). Dry weight at 60 8C (to constant weight) and total carbon and nitrogen (determined as percentage of sample dry weight on duplicate finely ground samples using a Carlo-Erba analyzer) were analyzed from the algae tissue. Total P content was determined after acid digestion by colorimetry (Jackson, 1970). The net growth rate (m) of C. linum was calculated following the equation: m = (ln Btln B0)t1, where Bt and B0 are the biomasses (dry weight) of the algae at the end and the beginning of the experiment, respectively, and t is the number of days. 2.3. Photosynthesis versus irradiance measurements In previous studies, we have observed that after 15 and 60 h of fertilization with phosphorus and nitrogen, respectively, almost all the dissolved inorganic phosphorus (from 17 to 0.8 mM) and nitrogen (from 100 to 15 mM) added to the jars with C. linum was absorbed (Mene´ ndez et al., 2002). Photosynthesis and dark respiration of C. linum were measured three times: (1) 3 days after the first fertilization, during the depletion period to measure the effect of nutrient storage in macroalgae; (2) 3 h after the second fertilization; and (3) 1.5 days after the second fertilization. In control jars, photosynthesis and respiration were measured on the same days as the rest of the treatments, but owing that were not fertilized, measurements corresponding to 3, 4 and 4.5 days without fertilization. Measurements were made as oxygen changed in filtered (0.45 mm mesh size) lagoon water (initial pH 8.94 0.029 and DIC 3.01 0.0046 mmol L1) in 110 mL stoppered bottles containing about 0.5 g fresh weight (0.03 g dry weight) of whole algal thalli (McGlathery et al., 1996). The bottles were placed in an incubator at a controlled temperature (20 8C), and stirring was performed by stirring bars (ca. 200 rpm). Daylight fluorescent tubes were used and the irradiance was adjusted by covering the bottles with neutral filters to assess photosynthesis–irradiance relationships (between 0 and 830 mmol photon m2 s1, 400– 700 nm). After 1.5 h of incubation, dissolved oxygen concentrations were measured to the nearest 0.01 mg using an oxygen electrode (MultiLine P3 WTW instruments) and checked with oxygen concentrations in bottles (n = 3) without algae. The alga was extracted of each bottle and was dried to constant weight at 70 8C. All the results were expressed in mg O2 g1 dry weight h1. Parameters describing the photosynthesis versus irradiance response (P–I) were determined for each experimental condition using a Sigmaplot computer program by fitting the observed data to the equation (Webb et al., 1974): P ¼ Pm ð1 expaI Þ þ R where P is gross photosynythesis, Pm is the maximum rate of net photosynthesis, a is initial slope representing photosynthetic efficiency at low light, I is irradiance, and R is respiration rate. 2.4. Statistical analysis The effects of nutrient enrichments on nutrient and chlorophyll content of the algae and on growth rate were statistically tested using one-way ANOVA. Data were log transformed
M. Mene´ ndez / Aquatic Botany 82 (2005) 181–192
185
Fig. 1. Nitrogen and phosphorus contents as percentage of dry weight in C. linum 0 day (represented by lines) and at 9 days (represented by bars) of the incubation period. Mean of three replicates and standard errors are reported. An asterisk represents significant differences with the control and between the beginning and the end of the experiment at the p < 0.05 level.
to obtain homogeneity of variances and determined to be normally distributed (Lilliefors test) before the analyses were conducted. Post hoc comparisons were made among treatment means using Tukey’s test, when ANOVA demonstrated that significant differences existed. To test the effect of enrichment on P–I curves, Student’s (paired) t-test (Legendre and Legendre, 1998) were used with a level of significance at 5%. The CSSStatistica computer program was used for statistical analysis.
3. Results 3.1. Algal content of N, P and C Enrichment with N alone, or in combination with P, resulted in significant increase (F = 53.8, Tukey’s test, p < 0.0005) of the N content of C. linum (Fig. 1), while enrichment with P alone had no effect (Tukey’s test, p > 0.05) on tissue-N. Enrichment with P alone or in combination with N, resulted in significant (F = 93.36, Tukey’s test, p < 0.0005) increases in the P content of C. linum, and this increase was higher in the treatments fertilized with P alone than in those treatments where both P and N, either as nitrate or ammonium, were added (Fig. 1). N-enrichments alone had no effect (Tukey’s test, p > 0.05) on the P content of C. linum. N enrichment increased the N:P ratio up to 21 in both nitrate plus P, and ammonium plus P treatments, and to 192–194 under N-alone enrichments as ammonium or nitrate related to the P-alone treatment (Table 1). A significant increase in carbon content was observed in the tissue of C. linum in the control jars (no nutrient additions) after 9 days (F = 3.05, Tukey’s test, p < 0.05). 3.2. Chlorophyll content of the algae N-enrichment was followed by an increase in the chlorophyll a and b content in the tissue of algae (F = 5.87, p < 0.01), and this increase was higher in the jars enriched with
186
M. Mene´ ndez / Aquatic Botany 82 (2005) 181–192
Table 1 Atomic N:P, C:N, C:P ratios and growth rates (m, day1) calculated from C. linum between 0 and 9 days
Initial NO3 NH4+ PO43 NH4+ + PO43 NO3 + PO43 Control
N:P ratio
C:N ratio
C:P ratio
m (day1)
63 0.2 194 11.4 192 53.5 6 0.9 21 4.2 21 0.2 82 0.2
32.9 2.15 19.7 0.03 19.5 0.79 41.8 5.16 19.6 0.63 20.9 1.83 47.6 0.01
3141 32 3780 46 4545 89 249 20 493 11 437 21 3809 40
– 0.130 0.002 0.114 0.004 0.101 0.001 0.110 0.007 0.128 0.007 0.088 0.001
Values are the mean calculated from algae in the three recipients used for each treatment (S.E.)
ammonium alone than in those enriched with nitrate alone or N, either ammonium or nitrate, plus P (Fig. 2). The chlorophyll a/b ratio was significantly lower in C. linum fertilized with ammonium plus P than in control jars. 3.3. Growth rates An increase of biomass was observed in all the treatments after 9 days of the experiment; the growth rate of C. linum was significantly higher in enriched jars than in controls (F = 12.15, p < 0.001) (Table 1). No significant differences were observed in growth rates between different fertilization treatments, except between C. linum enriched with nitrate and nitrate plus P related to growth rate obtained in P-alone treatment (Tukey’s test, p < 0.05). 3.4. Photosynthesis nutrients relationships A significant effect was observed in C. linum photosynthesis under N fertilization, both when it was added alone and in combination with P in the incubation performed after the second fertilization (t > 2.5, p < 0.05, eight comparisons: all treatments with nitrogen versus CT, after 3 h and 1.5 days). No significant differences were observed
Fig. 2. Concentration of total (a + b) chlorophyll and chlorophyll a/b ratios in C. linum at the end of the experiment (9 days). Mean of three replicates and standard errors are reported. An asterisk represents significant differences with the control at the p < 0.05 level.
M. Mene´ ndez / Aquatic Botany 82 (2005) 181–192
187
between incubations made after 3 h and 1.5 days of fertilization with both types of N alone, but the photosynthetic response was higher when nitrogen was added as ammonium than as nitrate (t > 2.2, p < 0.05, two comparisons: ammonium versus nitrate alone treatments after 3 h and 1.5 days). If N was added in combination with P, significant differences in photosynthesis were observed after 3 h and 1.5 days of fertilization when N was in the form of ammonium (t = 3.8, p < 0.005, one comparison: ammonium plus P after 3 h versus 1.5 days), but not when N was added as nitrate. Inhibition of the photosynthesis was observed only in the incubation made with C. linum during the depletion period after 3 days of the first nitrate plus P fertilization. The maximum value of Pm (11.6 mg O2 g1 dry weight h1) was observed after 3 h of fertilization with ammonium plus P (Table 2). No significant differences in photosynthesis were observed between enrichment treatments and controls during the depletion period (after 3 days of the first fertilization) (Table 2). Addition of P had a significant effect increasing the photosynthesis after 1.5 days of the second fertilization in relation with the control after 4.5 days (3 + 1.5 days) without P addition (t > 3.6, p < 0.005). In the jars without enrichment (CT jars), a significant decrease in photosynthesis was observed after 5 days (t > 5.5, p < 0.0005, two comparisons: CT after 3 and 4 days versus CT after 4.5 days). The addition of nitrate alone increased C. linum respiration after 3 h of fertilization, but this effect was not observed after 1.5 days (Table 2). Increasing values of respiration were observed in C. linum enriched with P alone or in combination with nitrate or ammonium; this effect was higher when N was added as ammonium. Fertilization with ammonium alone had no effect on respiration rate. Nutrient enhancement of photosynthetic efficiency under low light (a) was observed in all the jars fertilized. This increase was higher under ammonium plus P treatment (Table 2). An inverse relationship was observed between a and chlorophyll a/b ratio (r2 = 0.86, p < 0.05) (Fig. 3a).
4. Discussion From the results obtained in this experiments, both photosynthesis and growth of C. linum from Tancada lagoon were limited by N and P, because both added nutrients, alone or in combination, enhanced the Pm and growth rates. In fact, considering both Pm and growth rates, one finds that N has a major effect with respect to P. The atomic N:P ratio of the algae in the treatments with P alone (6.0) was below the ratio of 36 reported by Atkinson and Smith (1983), indicating deficiency of N relative to P. The C:P ratio was five to eight times above the value of 550 (Atkinson and Smith, 1983) in all the treatments without P, showing deficiency of P (Lapointe, 1997). However, availability of N and P would be very variable along all the annual cycle in the field; consequently physiological and morphological responses of the macroalgae could be very different under different environmental conditions. The growth rate ranged between 0.087 and 0.130 day1, which is within the range of growth rates reported for C. linum measured in laboratory (0.03–0.15 day1; McGlathery and Pedersen, 1999) and in situ (0.01–0.22 day1; Pedersen and Borum,
188
NH4+ NO3 PO43 NH4+ + PO43 NO3 + PO43 CT a b
Pm (mg O2 g1 dry weight h1)
R (mg O2 g1 dry weight h1)
3 days
3h
1.5 days
3 days
2.96 3.27 2.38 6.90 4.72 2.33
7.19 6.08 3.11 11.61 7.48 2.71
7.66 5.48 3.88 9.78 6.25 1.53
0.73 0.92 0.66 0.85 0.62 0.25
(0.34) (0.47) (0.24) (0.85) (1.14) (0.39)
(0.71) (0.54) (0.45) (1.01) (0.55) (0.49)a
4 days after the beginning of the experiment. 4.5 days after the beginning of the experiment.
(0.79) (0.64) (0.60) (0.75) (0.71) (0.21)b
3h (0.18) (0.23) (0.13) (0.47) (0.32) (0.21)
0.82 1.34 1.01 0.44 1.36 0.23
(0.38) (0.30) (0.23) (0.34) (0.30) (0.17)a
a (mg O2 g1 dry weight h1/mmol m2 s1) 1.5 days
3 days
3h
0.76 0.66 1.33 1.72 1.01 0.85
0.020 0.017 0.010 0.020 0.014 0.014
0.038 0.026 0.012 0.036 0.041 0.009
(0.42) (0.35) (0.33) (0.39) (0.38) (0.11)b
(0.0025) (0.0033) (0.0019) (0.0025) (0.0060) (0.0037)
1.5 days (0.0023) (0.0018) (0.0022) (0.0014) (0.0018) (0.0046)a
0.039 0.033 0.025 0.064 0.037 0.009
(0.0019) (0.0026) (0.0044) (0.0015) (0.0036) (0.0039)b
M. Mene´ ndez / Aquatic Botany 82 (2005) 181–192
Table 2 Maximum photosynthetic rate (Pm), respiration (R) and photosynthetic efficiency at low irradiances (a), and standard errors in parenthesis, estimated from the experimental incubations of C. linum with different nutrient additions, 3 days after the first fertilization (depletion period) and 3 h and 1.5 days after the second fertilization (see Section 2)
M. Mene´ ndez / Aquatic Botany 82 (2005) 181–192
189
Fig. 3. Relationship between Chlorophyll a/b ratio and a at the end of the experiment (a), and relationship between maximum photosynthetic rate (Pm) and photosynthetic efficiency under low light (a) over the range of tissue N levels (b).
1997). Some discrepancies were observed between growth rates and Pm. The highest photosynthetic rates were observed with ammonium plus P fertilization, but the highest growth rates occurred with nitrate and nitrate plus P fertilization. N is considered to be a limiting nutrient for primary production in temperate coastal ecosystems (Lobban and Harrison, 1994). Nitrate uptake occurs principally during the dark period, whereas, nitrate reductase activity is greatest during the day (Pedersen and Borum, 1997; Lopes et al., 1997). In this study, the nutrients were added during the light period, and most likely, uptake of ammonium was faster than nitrate uptake under light conditions. So, the results observed may be related to the fact that Pm is an instantaneous value of production in a short period of time (1.5 h) with light conditions, and growth rate reflected integrated production throughout the study (9 days), which incubated alternating light and dark conditions. Another explanation is that the cause of the larger Pm in the ammonium treatment results from a greater chlorophyll content which is consistent with the larger value for a in the ammonium compared to the nitrate treatment.
190
M. Mene´ ndez / Aquatic Botany 82 (2005) 181–192
4.1. Effect of enrichment in photosynthesis and chlorophyll concentration Increase of Pm in C. linum occurred rapidly in response to variations in the external N supply, but some differences were observed under treatments. These differences could be related to the fast utilization of P when N is available (Lapointe, 1997). In a previous study, after 15 h of C. linum fertilization (17 mM PO43), all the phosphorus dissolved in the water was absorbed (Mene´ ndez et al., 2002). Moreover, the highest a value (0.064 mg O2 g1 dry weight h1/mmol m2 s1) was obtained in the ammonium plus P treatment after 1.5 days of fertilization, suggesting a high efficiency of light use. In our study, inhibition of photosynthesis at light intensities higher than 300 mmol m2 s1 was observed only with nitrate plus P fertilization during the depletion period (after 3 days of the first fertilization). Photosynthesis may be inhibited by increased oxygen concentration, dissolved inorganic carbon depletion, and high pH and high light intensities (Falkowski and Raven, 1997). The absence of photosynthesis inhibition under ammonium plus phosphorus, in the same conditions that in the nitrate plus phosphorus experiments (pH variation from 8.96 to 9.56, 6% of DIC reduction in 1.5 h and the same irradiances), suggests that this may be in relation with the assimilation of nitrate stored in macroalgae by reduction catalized by nitrate reductase. In addition to carbon products, algal growth required reduced nitrogen (Behrenfeld et al., 2004). If based on nitrate, assimilation to an a-amino acid requires 10 electrons and one ATP that consumes a fraction of photosynthetic reductants associated with stimulated respiratory CO2 release in the light (Weger and Turpin, 1989). In this study, respiration rates increased immediately, following the addition of N, and this effect was more evident when N was added as nitrate. Probably, this increase in respiration rate is in relation to the reduction of nitrate and also in relation to supply of the carbon skeletons for protein synthesis (Turpin, 1991). N deficiency typically causes decrease in both photosynthesis pigments and Rubisco content/activity that correspond to marked declines in a (Falkowski and Owens, 1980) and Pm normalized to biomass (Turpin, 1991). In our study, a significant increase of chlorophyll was observed after N enrichment, and this effect was higher with ammonium enrichment showing N chlorophyll storage. Ammonium has a fast turnover rate into the algal cells, but little importance as a storage pool (McGlathery et al., 1996), whereas nitrate is an important nitrogen reserve pool in green macroalgae (Lopes et al., 1997; Naldi and Wheeler, 1999). This is probably the reason for a low increase in photosynthetic pigments when nitrate was added instead of ammonium as a source of N. Light-saturated photosynthesis and a (normalized to dry weight) were positively correlated over the range of tissue N levels (0.6–1.4% dry weight) (Fig. 3b), indicating that chlorophyll and Rubisco were equally limiting to photosynthesis, and that, light harvesting and C fixation were coupled (Falkowski and Owens, 1980). Photosynthetic efficiency at low irradiance (a) is regulated mainly by the effect of pigment level and configuration on light absorption and electron transport capacities (Turpin, 1991). In our study, an inverse relationship was observed between a and chlorophyll a/b ratio (Fig. 3a), showing that, when C. linum was acclimated to low irradiances (high values of a), a relatively high concentration of chlorophyll b related to chlorophyll a was observed. This structural modification leads to the absorption of longer wavelengths and allow the macroalgae to be more efficient under low light conditions.
M. Mene´ ndez / Aquatic Botany 82 (2005) 181–192
191
McGlathery and Pedersen (1999) suggested that a strategy to optimize both a and Pm may be a general characteristic of opportunistic thin-structured algae, such as C. linum, that represents a compromise between increasing light-harvesting and carbon-fixation capacities when the algae is exposed to variable light and nutrient regimes in estuarine environments. Correlations between a and Pm have been observed in Mediterranean marine macrophytes (Enriquez et al., 1996), macroalgae (Henley et al., 1991) and phytoplankton (Behrenfeld et al., 2004). This would be very beneficial for macroalgal growth in shallow coastal aquatic ecosystems, characterised by frequent wind events and nutrient pulsing discharges, that brings to high variability in irradiance and availability of nutrients.
Acknowledgements This work was supported by the research project ENV4-CT97-0584. European Commission.
References Atkinson, M.J., Smith, S.V., 1983. C:N:P ratios of benthic marine plants. Limnol. Oceanogr. 28, 567–574. Behrenfeld, M.J., Prasil, O., Babin, M., Bruyant, F., 2004. In search of a physiological basis for covariations in light-limited and light-saturated photosynthesis. J. Phycol. 40, 4–25. Enriquez, S., Duarte, C.M., Sand-Jensen, K., Nielsen, S.L., 1996. Broad-scale comparison of photosynthetic rates a cross phototrophic organisms. Oecology 108, 197–206. Falkowski, P.G., Owens, T.G., 1980. Light-shade adaptation: two strategies in marine phytoplankton. Plant Physiol. 66, 592–595. Falkowski, P.G., Raven, J.A., 1997. Aquatic Photosynthesis. Blackwell Science, Massachusetts, 375 pp. Henley, W.J., Levavasseur, G., Franklin, L.A., Osmond, C.B., Ramus, J., 1991. Photoacclimation and photoinhibition in Ulva rotundata as influenced by nitrogen availability. Planta 184, 235–243. Jackson, M.L., 1970. Ana´ lisis quı´mico del suelo. Omega, Barcelona, 662 pp. Lapointe, B.E., 1997. Nutrient theresholds for bottom-up control of macroalgal blooms on coral reefs in Jamaica and southeast Florida. Limnol. Oceanogr. 42, 1119–1131. Legendre, P., Legendre, L., 1998. Numerical Ecology, second ed. Elsevier, Amsterdam, 853 pp. Lobban, C.S., Harrison, P.J., 1994. Seaweed Ecology and Physiology. Cambridge University Press, Cambridge, 366 pp. Lopes, P., Oliveira, M.C., Colepicolo, P., 1997. Diurnal fluctuation of nitrate reductase activity in the marine red alga Gracilaria tenuistipitata (Rhodophyta). J. Phycol. 33, 225–231. Mene´ ndez, M., Comin, F.A., 2000. Spring and summer proliferation of floating macroalgae in a Mediterranean coastal lagoon (Tancada lagoon, Ebro Delta, NE Spain). Est. Coast. Shelf Sci. 51, 215–226. Mene´ ndez, M., Martı´nez, M., Comı´n, F.A., 2001. A comparative study of the effect of pH and inorganic carbon resources on the photosynthesis of three floating macroalgae species of a Mediterranean coastal lagoon. J. Exp. Mar. Biol. Ecol. 256 (1), 123–136. Mene´ ndez, M., Herrera, J., Comı´n, F.A., 2002. Effect of nitrogen and phosphorus supply on growth, chlorophyll content and tissue composition on the macroalga Chaetomorpha linum (O.F. Mu¨ ll) Ku¨ tz in a Mediterranean coastal lagoon. Sci. Mar. 66, 355–364. McGlathery, K.J., Pedersen, M.F., 1999. The effect of growth irradiance on the coupling of carbon and nitrogen metabolism in Chaetomorpha linum (Chlorophyta). J. Phycol. 35, 721–731. McGlathery, K.J., Pedersen, M.F., Borum, J., 1996. Changes in intracellular nitrogen pools and feedback controls on nitrogen uptake in Chaetomorpha linum (Chlorophyta). J. Phycol. 32, 393–401.
192
M. Mene´ndez / Aquatic Botany 82 (2005) 181–192
McGlathery, K.J., Krause-Jensen, D., Rysgaard, S., Christensen, P.B., 1997. Patterns of ammonium uptake within dense mats of the filamentous macroalga Chaetomorpha linum. Aquat. Bot. 59, 99–115. Naldi, M., Wheeler, P.A., 1999. Changes in nitrogen pools in Ulva fenestrata (Chlorophyta) and Gracilaria pacifica (Rhodophyta) under nitrate and ammonium enrichment. J. Phycol. 35, 70–77. Nixon, S.W., Owiat, C.A., Frithsen, J., Sullivan, B., 1986. Nutrients and the productivity of estuarine and coastal marine systems. J. Limnol. Soc. S. Afr. 12, 43–71. Peckol, P., DeMeo-Anderson, B., Rivers, J., Valiela, I., Maldonado, M., Weiner, J., Yates, J., 1994. Growth, nutrient uptake capacities, and tissue constituents of the macroalgae, Gracilaria tikvahiae and Cladophora vagabunda, related to site-specific nitrogen loading rates. Mar. Biol. 121, 175–185. Pedersen, M.F., Borum, J., 1997. Nutrient control of estuarine macroalgae: growth strategy and the balance between nitrogen requirements and uptake. Mar. Ecol. Prog. Ser. 161, 155–163. Ramus, J., Venable, M., 1987. Temporal ammonium patchiness and growth rate in Codium and Ulva (Ulvophyceae). J. Phycol. 23, 518–523. Ryther, J.H., 1989. Historical perspective of phytoplankton blooms on Long Island and the green tides of the 1950s. In: Cosper, E.M., Bricelj, V.M., Carpenter, E.J. (Eds.), Novel Phytoplankton Blooms. Coastal and Estuarine Studies Springer-Verlag, Berlı´n, pp. 375–381. Strickland, J.D., Parsons, T.R., 1972. A Practical Handbook of Seawater Analysis. Bulletin 167, second ed. Fisheries Research Board of Canada, Ottawa. Turpin, D.H., 1991. Effects of inorganic N availability on algal photosynthesis and carbon metabolism. J. Phycol. 27, 14–20. Valiela, I., Costa, J., Foreman, K., Teal, J.M., Howes, B., Aubrey, B., 1990. Transport of groundwater-borne nutrients from watersheds and their effects on coastal waters. Biogeochemistry 10, 177–197. Vergara, J.J., Bird, K.T., Niell, F.X., 1995. Nitrogen assimilation following ammonium pulses in the red algae Gracilariopsis lemaneiformis: effect on C metabolism. Mar. Ecol. Prog. Ser. 122, 253–263. Viaroli, P., Bartoli, M., Bondavalli, C., Naldi, M., 1995. Oxygen fluxes and dystrophy in a coastal lagoon colonized by Ulva rigida (Sacca di Goro, Po River Delta, northern Italy). Fresenius Environ. Bull. 4, 381–386. Vidal, M., 1994. Phosphate dynamics tied to sediment disturbances in Alfacs Bay (NW Mediterranean). Mar. Ecol. Prog. Ser. 110, 211–221. Vidal, M., Morguı´, J.A., 2000. Close and delayed benthic–pelagic coupling in coastal ecosystems: the role of physical constraints. Hydrobiology 429, 105–113. Webb, W.L., Newton, M., Starr, D., 1974. Carbon dioxide exchange of Alnus rubra: a mathematical model. Oecology 17, 281–291. Weger, H.G., Turpin, D.H., 1989. Mitochondrial respiration can support NO3 and NO2 reduction during photosynthesis, respiration, and N assimilationin the N-limited green algae Selenastrum minutum. Plant Physiol. 64, 828–832.